Thermal denaturation pathway of starch phosphorylase from Corynebacterium callunae: Oxyanion binding provides the glue that efficiently stabilizes the dimer structure of the protein

Abstract

Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 59-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures~Ն50 8C!, enzyme0oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of K sulfate ϭ 4.5 mM and K phosphate Ϸ 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by~a! an increase by about 5% in the relative composition of the a-helical secondary structure,~b! reduced 1 H0 2 H exchange, and~c! protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 59-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylasẽ probably due to tightened intersubunit contacts!, phosphate, and sulfate, this indirectly~1! preserves a functional active site up to Ϸ50 8C, and~2! stabilizes the covalent protein cofactor linkage up to Ϸ70 8C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 8C of Ϸ25 mM. The extra stability conferred by oxyanion-ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a Ϸ20 8C higher value on the temperature scale.Keywords: a-glucan phosphorylase; denaturation mechanism; oxyanion ligand; phosphate stabilization Unlike a great number of a-d-glycoside hydrolases, a-1,4-dglucan phosphorylases catalyze the degradation of a-1,4-d-glucan molecules by using phosphate rather than water as an acceptor of the transferred glucosyl moiety, as shown in Equation 1 where N is the degree of polymerization of the a-1,4-d-glucan.Therefore, interactions between the enzyme and the substrate phosphate play an essential role in the catalytic cycle of phosphorylase and contribute to binding energy in the ground state and the transition state of the reaction~Johnson et al

Thermal inactivation of D‐amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

“…A plausible scenario which receives additional support from previous evidence for the closely related DAO from Rhodotorula gracilis (Casalin et al, 1991) is that dissociation of the cofactor precedes the aggregation event and the aggregating species is the enzyme in its apo-form. As in other oligomeric enzymes displaying cofactor-dependent activity (e.g., Grießler et al, 2000; for a review on cofactor dissociation in flavoproteins, see Hefti et al, 2003), the functional dimeric state of D-amino acid oxidases is thought to be tightly linked to the bound FAD (Casalin et al, 1991;Piubelli et al, 2002). Therefore, cofactor release may involve dissociation of TvDAO subunits at some point.…”

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae

“…One unit of activity (1 U) refers to 1 µmol NADH produced under the given conditions. Binding of phosphate and sulfate to StP or mutants thereof was determined by using a previously reported procedure in which inhibition of quenching of cofactor fluorescence by iodide was measured [12]. CD spectroscopic measurements were carried out as described recently [12] using protein solutions (0.1 mg·mL −1 ± 10%) in a 20 m m Mops buffer, pH 7.0.…”

“…Binding of phosphate to a protein site different from the site where the substrate phosphate binds causes apparent tightening of quarternary interactions present in StP and leads to a 100‐fold increase in kinetic stability of the active dimer [12]. The very low in‐vitro lifetime of StP activity in the absence of phosphate (≈ 30 min [12]) suggests that this stabilizing effect might be important in the physiology of C. callunae . In light of the fact that interactions apparently critical to a stable and active protein conformation converge at the dimer interface of GP, we considered oxyanion‐dependent stability of the StP dimer to be a significant problem and hence worth examining.…”

Synthesis of branched polysaccharides with tunable degree of branching

“…On one hand the spectrum (B) is the same as the spectrum (A), which confirms the purity of the polysaccharide. On the other hand the spectrum (C) differs from the spectrum (A) by the peak in the amide I components band (1700-1600 cm −1 ) characteristic for the enzyme (Byler & Susi, 1986;Griessler, D'Auria, Tanfani, & Nidetzky, 2000), which confirms contamination of the polymer with the enzyme.…”